Method of cultivating bacteria proteins that are expressed in a temperature regulated manner

ABSTRACT

A method of cultivating bacteria having genes in plasmids which code for surface or membrane bound antigens or other proteins and which are expressed in a temperature regulated manner for the production of desired bacterial products, is disclosed. The bacteria are first cultivated in a culture medium to an inoculum under such temperature conditions that the bacteria retain their plasmids and no expression occurs, e.g. 20° C., and then in a culture medium under such temperature conditions that expression occurs and before the bacteria lose their plasmids they are harvested, and the desired product is isolated.

The present invention relates to a method of cultivating bacteria. Moreprecisely, the method is concerned with cultivating bacteria havinggenes in plasmids which code for surface or membrane bound antigens orother proteins and which are expressed in a temperature regulated mannerfor the production of desired bacterial products.

BACKGROUND

Bacteria and viruses often express on or within their membrane, proteinswhich in a certain environment function as a support for the organism toassociate in a specific way or adhere to a surface. Such a surface maybe the inner wall of the gastro-intestinal tract, the oesophagus or anyother biological surface or membrane in a human or animal body, where anorganism may find an optimal environment for growth. Membrane proteinsof this kind are often named colonization-factor-antigens (CFA) orfimbriae. In the literature, other words such as pili, hemagglutinins,filamentous or fibrillar proteins are used for the same kind ofsubstances. For convenience, "CFA" will be used herein to cover allthese kinds of membrane proteins which also may have an antigeniccharacter.

A number of bacteria which are of medical interest have been shown toproduce CFAs associated with their membrane. Among these, the ones whichare most important for humans, are organisms which colonize the humangastro-intestinal tract and the respiratory airways. Examples oforganisms which colonize the gastro-intestinal tract are enterotoxigenicEscherichia coli, Vibrio cholerae, Helicobacter pylor, Campylobacter,Shigellae, Salmonellae and Yersinia.

Of particular medical interest is the enterotoxigenic Escherichia coli(ETEC), since it is one of the major causes of diarrhea among childrenin the developing countries and accounts for more than 1 billiondiarrhea episodes and at least one million deaths per year, primarilyamong children. In the same way, Vibrio cholerae and Campylobacter causediarrhea among people in the developing countries. ETEC andCampylobacter are also the major cause of diarrhea among travellers tothese regions. Helicobacter pylori has recently become important due toits connection with stomach ulcers.

For most of these organisms the production of their CFAs is controlledby various factors in the environment, which in turn may activateregulatory genes in the plasmid DNA.

Thus, for a bacterium which has its natural growth environment in thehuman intestine it is advantageous to optimize its growth andpossibility of survival to conditions in the intestine. In other words,there is an optimal strategy of survival which implies the production ofadhesive proteins in the environment where there are suitable conditionsfor survival.

As is known from the literature, one such parameter that activatesregulatory plasmid genes is the temperature, implying that theproduction of CFAs in a number of bacteria is temperature regulated.Several human pathogenic organisms have been shown to produce CFAs attemperatures above room temperature only, and not at room temperature.Some of these organisms are of specific commercial interest, since ithas been shown that it is possible to produce oral vaccines againstthese organisms or bacteria.

In WO 92/14487 a method for production and use of an oral ETEC vaccineis disclosed. The ETEC bacteria are grown at 37° C. from agar plates toliquid medium in order to obtain commercial quantities of the ETECbacteria with CFAs and their sub-components (CS-antigens). Thesesubsequently formalin killed bacteria may then be used as an oralvaccine against the ETEC bacteria. The CFA and CS antigens will thusfunction as antigens in the immunological processing that will takeplace in the intestine.

In the scaling up of the production of ETEC bacteria having CFAs it wassurprisingly found that the bacteria lost their ability to produce CFAsmore and more for each new generation. In the studying of the reason forthis, it was noticed that the loss of the ability of the bacteria toproduce CFAs at temperatures above room temperature was accompanied by aloss of the regulatory gene localized in a plasmid in the bacteria.

DESCRIPTION OF THE INVENTION

The present invention is directed to a method of cultivating bacteriahaving genes in plasmids which code for surface or membrane boundantigens or other proteins and which are expressed in a temperatureregulated manner for the production of desired bacterial products,wherein

said bacteria are first cultivated in a culture medium to an inoculumunder such temperature conditions that the bacteria retain theirplasmids and no expression occurs, and then

said inoculum is further cultivated in a culture medium under suchtemperature conditions that expression occurs and before the bacterialose their plasmids they are harvested, and after that the desiredproduct is isolated.

Examples of genes in plasmids which code for other proteins and whichare expressed in a temperature regulated manner for the production ofdesired bacterial products are recombinant genes in plasmids whichretain their temperature regulated expression and produce desiredprotein products.

In a preferred embodiment, the first cultivation to an inoculum isconducted at room temperature and the further cultivation is conductedat the body temperature of a mammal.

In another preferred embodiment the room temperature is approximately20° C. and the body temperature of a mammal is 34-39° C.

In yet another preferred embodiment, the first cultivation to aninoculum is conducted in two steps, first on a culture plate and then ina liquid culture medium.

In still another preferred embodiment of the invention, the furthercultivation is conducted in a liquid culture medium.

These preferred embodiments of the invention make it possible tocultivate bacteria having genes in plasmids which code for surface ormembrane bound antigens or other proteins and which are expressed in atemperature regulated manner in large scale industrial fermentors forlarge scale production of desired bacterial products.

In an embodiment of the invention the cultivated bacteria areEscherichia coli expressing at least one type of colonization factorantigens selected from the group consisting of CFA/I, CS1, CS2, CS3,CS4, CS5 and CS6.

In such cultivation, the desired bacterial product may either be the E.coli carrying at least one of the colonization factor antigens CFA/I,CS1, CS2, CS3, CS4, CS5 and CS6 or such colonization factor antigensisolated from the bacteria.

EXAMPLES

In the following examples CFA medium has been used as culture medium.The composition of the liquid CFA medium is as follows: Casamino Acids1% (w/v), Yeast extract 0.078% (w/v), MgSO₄ 0.4 mM, MnCl₂ 0.04 mM, H₂ Odeionized, pH 7.4. As culture plates, CFA agar plates have been used.These include CFA medium with 20 g/liter added.

Example 1.

An ETEC stain expressing CS2+CS3 antigens was taken from -70° C. andevaluated regarding expression of the membrane components as follows.

CFA agar plates were inoculated with the strain CS2+CS3 and incubated at20° C. or 37° C. for 24 hours. The bacteria were harvested from theplates and samples containing 40×10⁶ bacteria each were furtherinoculated in a 800 ml (CFA medium) shake culture at 20° C. or 37° C.for another 24 hours. At 70 hours a sample of 4.5 ml was taken from the20° C. culture and was further cultivated at 37° C. in a 800 ml shakeculture (CFA medium). At various intervals the expression of CS2 wasanalyzed with an ELISA technique using a monoclonal antibody which hasbeen shown to have specificity for the CS2 antigen. All ELISA resultshave been adjusted to standardized amount of bacteria as measured bytheir optical density. The results as measured in ELISA-arbitrary unitsare presented in the Table 1.

                  TABLE 1    ______________________________________             ELISA units                        ELISA units   ELISA units    Time (hours)             at 20° C.                        at 20° C. + 37° C.                                      at 37° C.    ______________________________________    0        8          8             8    24       8          8             643    48       8          8             762    54       8          8             897    70       25         25            1127    74       58         361           814    79       64         736           537    96       85         850           214    ______________________________________

Example 2.

Under the same conditions as in Example 1, ETEC bacteria with CS4antigen were grown in liquid CFA medium. After the sampling at 31 hours,the temperature was raised to 37° C.

The results as measured in ELISA-arbitrary units can be seen in Table 2.

                  TABLE 2    ______________________________________                 ELISA units                           ELISA units    Time (hours) at 20° C.                           at 20° C. + 37° C.    ______________________________________    0            0         0    24           0         0    26           0         0    29           0         0    31           0         0    48           0         8    52           1         9    54           1         10    ______________________________________

Example 3.

Under the same conditions as in Example 1, ETEC bacteria with CS5antigen were grown in liquid CFA medium. After the sampling at 31 hours,the temperature was raised to 37° C.

The result as measured in ELISA-arbitrary units can be seen in Table 3.

                  TABLE 3    ______________________________________                 ELISA units                           ELISA units    Time (hours) at 20° C.                           at 20° C. + 37° C.    ______________________________________    0            0         0    24           0         0    26           0         0    29           0         0    31           0         0    48           1         5    52           1         8    54           1         8    ______________________________________

Example 4.

A culture of ETEC bacteria expressing the antigen CS1 was grownovernight in liquid CFA medium on a shaker at 37° C. Colonies wereplated on CFA agar and incubated at 37° C. overnight. The following day,colonies expressing CS1 fimbriae were detected by a colony blot methodusing a CS1 specific mouse monoclonal antibody as second antibody.Immunoblots were developed by a nitro blue tetrasodium salt stain.Individual CS1 fimbriae positive and negative colonies were picked andcultivated, spread on CFA agar plates as described above.

This time each plate was probed with one filter for the CS1 specificmonoclonal antibody as described above and also with one filter that wasused for colony hybridization with one of two radioactivity labelled(32P) probes. One probe specific for the structural gene of CS1 (cooA)and one probe specific for the regulatory RNS protein (rns).

Postive colonies: All colonies scored positive with the antibody assaywere also scored positive with the rns probe and CS1 probe.

Negative colonies: No colonies scored negative with antibody had anyreaction with the rns probe, a few negative colonies had lost also theirreactivity with the CS1 probe but most of them had retained their CS1structural genes.

In conclusion, there is a concomitant loss of CS1 antibody reactivity(expression of CS1 fimbriae) and loss of reactivity with the rns probe(loss of the plasmid encoding the rns gene).

We claim:
 1. A method of cultivating bacteria having genes in plasmidswhich code for surface or membrane bound antigens or other proteins andwhich are expressed in a temperature regulated manner for the productionof desired bacterial products wherein said plasmids are retained duringcultivation comprising:(a) inoculating and cultivating said bacteria ina culture medium at room temperature so that the bacteria retain theirplasmids and no expression occurs; (b) further cultivating said culturemedium of step (a) in a culture medium at 34-39° C. so that expressionof said surface or membrane bound antigens or other proteins occurs; (c)harvesting said bacteria before they lose their plasmids; and (d)isolating the desired bacterial products, said products being thesurface or membrane bound antigens or other proteins, or the bacteriahaving genes in plasmids which code for such surface or membrane boundantigens or other protein.
 2. The method of claim 1, wherein said roomtemperature is approximately 20° C.
 3. The method of claim 1, whereinsaid cultivating at room temperature of step (a) is conducted in twosteps, first on a culture plate and then in a liquid culture medium. 4.The method of claim 1, wherein the further cultivation is conducted in aliquid culture medium.
 5. The method of claim 1, wherein said bacteriaare Escherichia coli expressing at least one type of colonization factorantigens selected from the group consisting of CFA/l, CS1, CS2, CS3,CS4, CS5 and CS6.
 6. The method of claim 1, wherein said desiredbacterial product is at least one of the colonization factor antigensselected from the group consisting of CFA/I, CS1, CS2, CS3, CS4, CS5 andCS6.
 7. The method of claim 2, wherein said cultivating at roomtemperature of step (a) is conducted in two steps, first on a cultureplate and then in a liquid culture.
 8. The method of claim 2, whereinsaid further cultivating is conducted in a liquid culture medium.
 9. Themethod of claim 3, wherein said further cultivating is conducted in aliquid culture medium.
 10. The method of claim 2, wherein said bacteriaare Escherichia coli expressing at least one type of colonization factorantigens selected from the group consisting of CFA/l, CS1, CS2, CS3, CS5and CS6.
 11. The method of claim 3, wherein said bacteria areEscherichia coli expressing at least one type of colonization factorantigens selected from the group consisting of CFA/l, CS1, CS2, CS3, CS5and CS6.
 12. The method of claim 4, wherein said bacteria areEscherichia coli expressing at least one type of colonization factorantigens selected from the group consisting of CFA/l, CS1, CS2, CS3, CS5and CS6.